Norikatsu Kasuga

Eccentric exercise‐induced morphological changes in the membrane systems involved in excitation—contraction coupling in rat skeletal muscle

Physiological evidence suggests that excitation‐contraction (E—C) coupling failure results from eccentric contraction‐induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E—C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast‐twitch (FT) and slow‐twitch (ST) muscle fibres involved in E—C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E—C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles. Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor‐driven treadmill running (18 m min−1, 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca2+–K3Fe(CN)6–OsO4 techniques, and observed by high voltage electron microscopy (120–200 kV). There were four obvious ultrastructural changes in the arrangement of the transverse (t)‐tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t‐tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t‐tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2–3 days after the downhill running exercise. The eccentric exercise led to the formation of abnormal membrane systems involved in E–C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise‐ induced E–C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E–C coupling process in skeletal muscle.

Morphological changes in the triads and sarcoplasmic reticulum of rat slow and fast muscle fibres following denervation and immobilization

SummaryWe observed the morphological features of the membrane systems (sarcoplasmic reticulum, transverse tubules and triads) involved with the excitation-contraction coupling in rat soleus and extensor digitorum longus muscle following two disuse protocols: denervation and immobilization. The immobilized positions were: maximum dorsal flexor (soleus were stretched and extensor digitorum longus were shortened), maximum plantar flexor (soleus were shortened and extensor digitorum longus were stretched), and midway between the dorsal flexor and plantar flexor. The arrangement of the membrane systems was disordered following both disuse conditions. Increases in transverse tubule network were apparent; there were clearly more triads than in normal fibres, and pentadic and heptadic structures (i.e., a close approximation of two or three transverse tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum) were frequently appeared following both denervation and immobilization. The most notable difference between the influence of denervation and immobilization on the membrane systems is the time at which the pentads and heptads appeared. They appeared much earlier (1 week after denervation) in denervated than in immobilized (3 or 4 weeks after immobilization) muscle fibres. On the other hand, the frequency of pentads and heptads is clearly related to the fibre type (significantly higher in extensor digitorum longus) and to extent of atrophy. The different influences of immobilization in each leg position suggest that disuse, but with neurotrophic factor(s), influences on the membrane systems were affected by sarcomere length, and the neurotrophic factor(s) and muscle activity were not always necessary to form mew membrane systems in disuse skeletal muscle fibres.

Plasticity of the transverse tubules following denervation and subsequent reinnervation in rat slow and fast muscle fibres

We have studied the effects of short term denervation followed by reinnervation on the ultrastructure of the membrane systems and on the content of and distribution of key proteins involved in calcium regulation of fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus (SOL) muscle fibres. Ischiadic nerve freezing resulted in total lack of neuromuscular transmission for 3 days followed by a slow recovery, but no decline in twitch force elicited by direct stimulation. The latter measurements indicate no significant atrophy within this time frame. The membrane systems of skeletal muscle fibres were visualized using Ca2+-K3Fe(CN)6-OsO4 techniques and observed using a high voltage electron microscope. [3H]nitrendipine binding was used to detect levels of dihydropyridine receptor (DHPR) expression. The Ca2+ pumping free sarcoplasmic reticulum domains were not affected by the denervation, but the Ca2+ release domains were dramatically increased, particularly in the FT-EDL muscle fibres. The increase is evidenced by a doubling up of the areas of contacts between SR and transverse (t-) tubules, so that in place of the normal triadic arrangement, pentadic and heptadic junctions, formed by multiple interacting layers of ST and t-tubules are seen. Frequency of pentads and heptads increases and declines in parallel to the denervation and reinnervation but with a delay. Immunofluorecence and electron microscopy observations show presence of DHPR and ryanodine receptor clusters at pentads and heptads junctions. A significant (P > 0.01) positive correlation between the level of [3H]nitrendipine binding component and the frequency pentads and heptads was observed in both the FT-EDL and ST-SOL muscle fibres indicating that overexpression of DHPRs accompanies the build up extra junctional contacts. The results indicate that denervation reversibly affects the domains of the membrane systems involved in excitation-contraction coupling.

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres

SummaryThe deteriorating effect of μm order of Ca2+ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca2+ in quantities of more than 0.1 μm. Low temperature was seen to suppress (Q10>4), and neutral pH to maximize, this decrease in tension. In rigor solution containing Ca2+, SDS electrophoresis indicated that a 95 k dalton component (α-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.

The expression patterns of Pax7 in satellite cells during overload-induced rat adult skeletal muscle hypertrophy.

Aim:  Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self‐renewal. Because the increase in the SC population is induced during overload‐induced skeletal muscle hypertrophy, it is possible that Pax7‐regulated SC self‐renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood.

Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop; however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.

Differential response of the membrane systems involved in excitation-contraction coupling to early and later postnatal denervation in rat skeletal muscle.

We compared the morphological features of the membrane systems involved in excitation–contraction (E–C) coupling during early postnatal development stages in rat skeletal muscles (tibialis anterior) denervated either at birth or 7 days after birth. Four obvious structural changes are observed in the arrangement of the transverse (T) tubule network and the disposition of triads following early postnatal denervation: (1) an increase in the longitudinal segments of the T tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolae clusters, (4) the appearance of pentads and heptads (i.e. a close apposition of two or three T tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum). The increased presence of longitudinal T tubules parallels the loss of cross striations, and this in turn is due to misalignment of the myofibrils. The clusters of caveolae appear almost exclusively in muscle fibres denervated at birth, and pentads and heptads are more frequently observed in muscles denervated at 7 days. The differential growth of muscle fibres in response to denervation leads to the formation of abnormal membrane systems involved in the E–C coupling with very unique morphological features, which differ from the case of denervation in adult muscle fibres.

Influences of sarcomere length and selective elimination of myosin filaments on the localization and orientation of triads in rat muscle fibres

SummaryUltrastructural features of internal membrane systems directly concerned with the excitation-contraction coupling were observed in chemically skinned muscle bundles prepared from Wistar rat extensor digitorum longus muscle to clarify two questions: (1) whether triads localization and orientation are influenced by the sarcomere length and (2) whether triads localization and orientation are influenced by the selective elimination of myosin filaments. The distance between triads and Z-lines depends on the sarcomere length: it increase with sarcomere length. There is a highly significant (p<0.01) positive correlation between sarcomere length and the distance between triads and Z-line. The distance between Z-line and triads is dependent on sarcomere length, but the width of junctional gap remains constant when the sarcomere length was changed. Incubation in a concentration of KCl, which dissolves the myosin filaments. The localization and orientation of triads was not altered by the elimination of myosin filaments, however, the distance between the Z-line and triads becomes shorter when the myosin filaments was completely eliminated. There were significant differences (p<0.01) between control and myosin filament eliminated fibres in the distances between Z-lines and triads (over 2 μm). These results indicate that the distance between triads and Z-lines depend on the sarcomere length and that there may be some connection(s) between triads and the myofibrils. There is that the elastic component responsible for tethering the triads in their normal position is interrupted either because it is normally attached to the myosin filaments, or because it is extracted by the conditions that dissociate myosin filaments.

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.

In Vivo Real-Time Imaging of Exogenous HGF-Triggered Cell Migration in Rat Intact Soleus Muscles.

The transplantation of myogenic cells is a potentially effective therapy for muscular dystrophy. However, this therapy has achieved little success because the diffusion of transplanted myogenic cells is limited. Hepatocyte growth factor (HGF) is one of the primary triggers to induce myogenic cell migration in vitro. However, to our knowledge, whether exogenous HGF can trigger the migration of myogenic cells (i.e. satellite cells) in intact skeletal muscles in vivo has not been reported. We previously reported a novel in vivo real-time imaging method in rat skeletal muscles. Therefore, the present study examined the relationship between exogenous HGF treatment and cell migration in rat intact soleus muscles using this imaging method. As a result, it was indicated that the cell migration velocity was enhanced in response to increasing exogenous HGF concentration in skeletal muscles. Furthermore, the expression of MyoD was induced in satellite cells in response to HGF treatment. We first demonstrated in vivo real-time imaging of cell migration triggered by exogenous HGF in intact soleus muscles. The experimental method used in the present study will be a useful tool to understand further the regulatory mechanism of HGF-induced satellite cell migration in skeletal muscles in vivo.